Mechanical stretch induces nerve sprouting in rat sympathetic neurocytes
Introduction
Sympathetic nerve sprouting (SNS) is a major factor for the development of malignant arrhythmias after myocardial infarction (MI) (Zhou et al., 1995) and congestive heart failure (HF) (Himura et al., 1993). Pathological outgrowth of sympathetic fibers and subsequent hyperinnervation seems to be an important contributor to an elevated sympathetic tone and has been reported to occur within the first hours of MI (Oh et al., 2006). Furthermore, SNS has been suggested to be a major contributor to sudden cardiac death (Cao et al., 2000a, Cao et al., 2000b). After MI increased nerve growth factor (NGF) expression has been shown to promote sympathetic hyperinnervation (Hasan et al., 2006), and may represent an adaptive mechanism by which the heart tries to maintain ventricular contractility at the cost of eventually triggered ventricular arrhythmias. There is evidence that inflammatory cells like myofibroblasts and macrophages contribute to the development of SNS in the ischemic injury area after MI (Hasan et al., 2006). If other contributing factors than inflammatory cells or NGF are involved in the pathogenesis of SNS still needs to be investigated. Mechanical stretch is such condition to which the cardiac cells are exposed after MI and during HF (Zimmerman et al., 2000, Ashikaga et al., 2005, Gao et al., 2007, Koba et al., 2008). Recently, we could demonstrate that mechanical stretch of ventricular cardiomyocytes induces a significant down-regulation of NGF (Rana et al., 2009). However, a stretch-induced NGF down-regulation in cardiomyocytes would not explain the induction of SNS after MI and HF. Because the myocardium in extensively innervated by the sympathetic nervous system and the impact of mechanical stretch on sympathetic neurocytes is unknown, this study aimed to investigate the impact of mechanical stretch on rat neonatal sympathetic neurocytes of the superior cervical ganglion (SCG). Furthermore, stretch-induced alterations of the neurotrophins NGF, neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), glial derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) were investigated. Mechanical stretch resulted in increased neural growth assessed by measuring the neurite outgrowth and growth associated protein 43 (GAP-43) expression. Furthermore, in the cell culture supernatant of neurocytes exposed to mechanical stretch NGF and CNTF expression was significantly increased, GDNF expression was down-regulated, NT-3 expression was unchanged, while BDNF could not be detected. By anti-neurotrophin neutralizing experiments, NGF and CNTF were identified as important stretch-induced neural growth-inducing factors. Angiotensin-II type 1 (AT-1) receptor blockade with losartan completely prevented the stretch-induced neural growth by attenuating the stretch-induced increase of NGF and CNTF.
Section snippets
Superior cervical ganglia (SCG) neurocyte cell culture
All animals were cared for according to institutional animal-care requirements. Primary cultures of SCGs were performed from postnatal day 1 to 3 Sprague Dawley rats (Charles River, Germany) as described previously (Rana et al., 2009). Briefly, both SCG of 20–30 neonatal rat were dissected from the carotid bifurcation, cut into small pieces, and then transferred into 1 ml of Neuronal Base Medium (PAA, U15-024) supplemented with collagenase II (1.0 mg/ml, 280 U/mg, Biochrom, C2-22), trypsin (0.5
Mechanical stretch induces growth of neonatal rat sympathetic neurocytes
Mechanical stretch of SCG neurocytes over a time period of 72 h resulted in a significant increase of neurite outgrowth compared to control neurocytes (in µm: control: 0 h = 41.9 ± 1.3 vs. 72 h = 49.4 ± 2.4, p = 0.13, n = 3 preparations; stretch: 0 h = 45.1 ± 0.9 vs. 72 h = 71 ± 1.7, p < 0.01, n = 3 preparations) (Fig. 1A). The net increase of neurite outgrowth of stretched neurocytes after 72 h was significantly increased compared to un-stretched control neurocytes (control: 7.5 ± 1.7 vs. stretch: 25.9 ± 1.3, n = 3 preparations,
Discussion
This study demonstrates: i) cellular stretch leads to an increased growth of rat sympathetic neurocytes; ii) neonatal rat SCG neurocytes express NGF, CNTF, NT-3, GDNF, but not BDNF mRNA and protein; iii) the stretch response is coupled to an increase of NGF and CNTF, while NT-3 remains unaffected, GDNF is down-regulated and BDNF could not be detected; iv) by specific neurotrophin neutralizing experiments, NGF and CNTF were identified as important stretch-induced neural growth-inducing factors;
Acknowledgements
We thank Esra Saygili for her helpful assistance. This work contains data from the doctoral thesis of Dorothee Hommes (University RWTH Aachen, Germany).
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